Abstract
Human endometrium is a high dynamic tissue that contains endometrial stromal stem cells (hESSCs). The hESSCs have been differentiated into a number of cell lineages. However, differentiation of hESSCs into megakaryocytes (MKs) has not yet been investigated. The aim of this study was to investigate the feasibility of MK generation from hESSCs and subsequent production of functional platelets (PLTs). In our study, hESSCs were cultured from endometrial stromal cells as confirmed by positive stromal cell specific markers (CD90 and CD29) and negative hematopoietic stem cell markers (CD45 and CD34) expression. Then, hESSCs were differentiated in a medium supplemented with thrombopoietin (TPO) for 18 days. The MK differentiation was analyzed by flow cytometry and confocal microscopy. The differentiation medium was collected for PLT production analysis by flow cytometry, transmission electron microscopy and functional measurements. Our results show: 1) MKs were successfully generated from hESSCs as identified by expression of specific markers (CD41a: 1±0.09% and 39±3.0%; CD42b: 1.2±0.06% and 28±2.0%, control vs. differentiation) accompanied with reduction of pluripotent transcription factors (Oct4 and Sox2) expression; 2) The level of PLTs in the differentiation medium was 16±1 number/µl as determined by size (2–4 µm) and CD41a expression (CD41a: 1±0.4% and 90±2.0%, control vs. differentiation); 3) Generated PLTs were functional as evidenced by the up-regulation of CD62p expression and fibrinogen binding following thrombin stimulation; 4) Released PLTs showed similar ultra-structure characteristics (alpha granules, vacuoles and dense tubular system) as PLTs from peripheral blood determined by electron microscopic analysis. Data demonstrate the feasibility of generating MKs from hESSCs, and that the generated MKs release functional PLTs. Therefore, hESSCs could be a potential new stem cell source for in vitro MK/PLT production.
Highlights
Platelets (PLTs) play a key role in haemostatic plug formation [1] and arterial thrombosis [2]
Characterization of Human endometrial stromal stem cells (hESSCs) The passage 4 and 6 (P4, 6) cultured cells were characterized by flow cytometry and immunocytochemistry methods
Flow cytometric results showed that the hESSCs positively expressed stromal cell specific markers CD90 and CD29 (Fig 1-D, E) and negatively stained with hematopoietic lineage markers CD45 and CD34 (Fig 1-F, G)
Summary
Platelets (PLTs) play a key role in haemostatic plug formation [1] and arterial thrombosis [2]. PLT transfusion is a mainstay therapy of various PLT-related diseases, such as thrombocytopenia and in cases of menorrhagia that are refractory to medical therapy [3,4,5,6]. In the United States, the total number of PLT transfusions is over 10 million units per year and is increasing annually. Traditional source of PLTs from blood donors has several limitations, including short storage life of PLT concentrates and risk of pathogenic contamination [7,8]. Developing a reliable, safe alternative approach to producing large numbers of PLTs is intensely appealing. Stem cell based generation of megakaryocytes (MKs), the parent cells of circulating PLTs, would provide significant advantages over the current donor-supply approach. There are several wellestablished limitations of these sources, including achieving adequate amounts of HSCs and flawed maturation, as well as political and ethical ramifications
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